Antibacterial Activity of Pseudonocardia Sp. JB05, a Rare Salty Soil Actinomycete Against Staphylococcus Aureus

Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 182945, 7 pages http://dx.doi.org/10.1155/2014/182945 Research Article Antibacterial Activity of Pseudonocardia sp. JB05, a Rare Salty Soil Actinomycete against Staphylococcus aureus Nesa Jafari,1,2 Reza Behroozi,1 Davoud Farajzadeh,3,4 Mohammad Farsi,5 and Kambiz Akbari-Noghabi1 1 Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology, Tehran, Iran 2 Department of Plant Biotechnology, Buali-Sina University of Hamedan, Iran 3 Biotechnology Research Center, Biomedicine Institute, Tabriz University of Medical Sciences, Tabriz, Iran 4 Department of Cellular and Molecular Biology, Faculty of Biological Sciences, Azarbaijan Shahid Madani University, Tabriz, Iran 5 Department of Plant Biotechnology, Ferdowsi University of Mashhad, Mashhad, Iran Correspondence should be addressed to Davoud Farajzadeh; [email protected] Received 29 May 2014; Revised 9 July 2014; Accepted 13 July 2014; Published 14 August 2014 Academic Editor: Medicharla V. Jagannadham Copyright © 2014 Nesa Jafari et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Staphylococcus aureus is a Gram-positive bacterium that causes many harmful and life-threatening diseases. Some strains of this bacterium are resistant to available antibiotics. This study was designed to evaluate the ability of indigenous actinomycetes to produce antibacterial compounds against S. aureus and characterize the structure of the resultant antibacterial compounds. Therefore, a slightly modified agar well diffusion method was used to determine the antibacterial activity of actinomycete isolates against the test microorganisms. The bacterial extracts with antibacterial activity were fractionated by silica gel and G-25 sephadex column chromatography. Also, the active fractions were analyzed by thin layer chromatography. Finally, the partial structure of the resultant antibacterial compound was characterized by Fourier transform infrared spectroscopy. One of the isolates, which had a broad spectrum and high antibacterial activity, was designated as Pseudonocardia sp. JB05, based on the results of biochemical and −1 16S rDNA gene sequence analysis. Minimum inhibitory concentration for this bacterium was 40 AU mL against S. aureus.The antibacterial activity of this bacterium was stable after autoclaving, 10% SDS, boiling, and proteinase K. Thin layer chromatography, using anthrone reagent, showed the presence of carbohydrates in the purified antibacterial compound. Finally, FT-IR spectrum of the active compound illustrated hydroxyl groups, hydrocarbon skeleton, and double bond of polygenic compounds in its structure. To the best of our knowledge, this is the first report describing the efficient antibacterial activity by a local strain of Pseudonocardia. The results presented in this work, although at the initial stage in bioactive product characterization, will possibly contribute toward the Pseudonocardia scale-up for the production and identification of the antibacterial compounds. 1. Introduction areadmittedtoAmericanhospitalsannuallyduetostaphylo- coccal infection [1]. Penicillin is a common medication used Staphylococcus aureus (S. aureus) is a facultative anaerobic, to treat an S. aureus infection. However, the problem of peni- Gram-positive coccus. This bacterium is the most common cillin resistance is extremely pronounced in most countries, cause of a wide variety of illnesses, such as impetigo, pimples, where first-line therapy is most commonly a penicillinase- boils (furuncles), cellulitis folliculitis, carbuncles, scalded resistant -lactam antibiotic such as oxacillin or flucloxacillin skin syndrome, and abscesses, as well as life-threatening [2]. Soil bacteria belonging to actinomycetes group are diseases, such as pneumonia, osteomyelitis, meningitis, endo- oneofthebestsourcesofbioactivenaturalcompounds carditis, toxic shock syndrome (TSS), bacteremia, and sepsis. used as antibiotics, pesticides, pharmaceuticals, herbicides, It can affect almost any parts of the body, including skin, antiparasitics, and enzymes [3, 4]. About 13,000 biologically connective tissue, respiratory, bone, joint, and endovascular active secondary metabolites have been discovered from regions. One estimate indicated that nearly 500,000 patients actinomycetes, and from which 70% have already been 2 BioMed Research International purified [5]. Within actinomycetes, Streptomyces is the most MgSO4⋅7H2O, 0.05 g; CaCO3, 0.02 g; FeSO4⋅7H2O, 0.01 g, investigated genus because of either commercial interests or and agar 15.0 g, pH 8, Czapek Dox Agar (Sucrose, 30.0 g; dominanceofthegenusondilutionplatesanditsfacilityof NaNO3 3.0 g; K2HPO4 1.0g;KCl0.5g;MgSO4⋅7H2O, 0.5 g; isolation [6]. FeSO4⋅ 7H2O, 0.01 g; and agar 15.0 g; pH 7), and ISP2 (Malt It has been found that among actinomycetes the fre- extract, 10.0 g; Yeast extract, 4.0 g; Glucose, 4.0 g, and agar quencies of isolation for Streptomyces, Actinoplanes, Actino- 20.0 g; pH 7) were used for identification, isolation, and madura, Microbispora, Micromonospora, Nocardia, Pseudo- preservation of actinomycetes. All chemical reagents and nocardia, Streptosporangium, Thermoactinomyce, and Ther- solvents were provided from Merck, Germany. momonospora were 95.3, 0.2, 0.1, 0.18, 1.4, 1.98, 0.06, 0.10, and To provide antibacterial compounds secreted out into the 0.14%, respectively. culture medium, actinomycete isolates were cultured into Non-Streptomycete actinomycetes (NSA) are a group ISP2 broth medium and incubated on a rotary shaker at ∘ ofactinomycetesthatwerereviewedbyEl-Tarabilyand 160 rpm for 14 days at 30 C. Sivasithamparam [6]. H. A. Lechevalier and M. P.Lechevalier Test bacteria, including Staphylococcus aureus, Bacillus (1967) isolated 5000 actinomycetes from the soil and found subtilis,B.pusteurii,Escherichiacoli,Klebsiellasp., Pseu- that the genera of NSA, which were evaluated as rare, domonas aeruginosae, Xanthomonas sp., and Acetinobacter Thermomonospora, Actinoplanes, Microbispora, Thermoacti- sp., provided from the Persian Type Culture Collection of nomyces, Streptosporangium, Micropolyspora, Pseudonocar- the Iranian Research Organization for Science and Technol- dia, and Microellobosporia,formlessthan0.2%ofthetotal ogy (IROST) (http://portal.irost.org/persian/PTCC/), were ∘ isolates [7]. grown overnight in Luria-Bertani (LB) medium at 30 C. The family of Pseudonocardiaceae contains seven gen- The preliminary identification of isolates were carried out era (Actinopolyspora, Amycolata, Amycolatopsis, Kibdelospo- based on the result of biochemical conventional tests analysis rangium, Pseudonocardia, Saccharomonospora, and Saccha- [12]. Morphology and Gram stain were also determined by ropolyspora)[8],severalofthemwithindustrialinterestin the use of a light microscope (1,000x) (Zeiss, Argentina S.A). bioconversion processes as antibiotic producers, including erythromycin, vancomycin, and rifamycin [9]. Li et al. (2011) 2.2. Antibacterial Activity Assay. Aslightlymodifiedagar reported the production of three new diazaanthraquinone well diffusion method [13] was adopted to determine the derivatives by the strain SCSIO 01299, a marine actinomycete antibacterial activity of twenty-six actinomycete isolates member of the genus Pseudonocardia,isolatedfromdeep- against the test microorganisms with three replicates. Briefly, sea sediment of the South China Sea. They found that some eight millimeter wells were made in Muller Hinton agar of these compounds exhibited potent cytotoxic activities plates and inoculated with the test microorganisms. The same against three tumor cell lines (SF-268, MCF-7, and NCI- concentration of the diluted extracts of each actinomycete H460) with IC50 values between 0.01 and 0.21 Mand isolates then was poured into the wells. After 15-, 24- and also showed antibacterial activities against Staphylococcus ∘ 48-hour incubation at 28 C, the antibacterial activity of aureus ATCC 29213, Enterococcus faecalis ATCC 29212, and isolates was evaluated by measuring the diameter of the clear Bacillus thuringensis SCSIO BT01, with minimum inhibitory −1 inhibition zones around the wells. The minimum inhibitory concentration (MIC) values of 1–4 gmL [10]. concentrations (MICs) were determined using the microplate As it was referred to earlier, actinomycetes produce dilution method. many useful antibiotics against a large number of pathogenic The protein concentration of cell lysates was determined bacteria. In the last decade, the screening for new secondary by the Bradford’s method [14]. metabolites with antibacterial activity has also focused on minor groups of actinomycetes, including species that are difficult to isolate and culture, and those that grow under 2.3. Preparation of DNA and Amplification of 16S rDNA extreme conditions (i.e., alkaline and acidic conditions) [11]. Gene. DNA was prepared using a DNA extraction kit Therefore, this study was designed to evaluate the ability of (QIAamp DNA Mini Kit, USA) and 16S rRNA encod- indigenous actinomycete strain(s) to produce antibacterial ing gene, amplified using primers S-C-Act-235-S-20 (5 - compound(s) against S. aureus in order to identify potent CGCGGCCTATCAGCTTGTTG-3 ) and S-C-Act-878-A-19 antibacterial compound(s). (5 -CCGTACTCCCCAGGCGGGG-3 )[15]. PCR reactions were carried out in 25 L reaction

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